Total joint replacement (TJR) is a highly successful surgical procedure; however the long-term survivorship is limited by wear of the bearing surfaces. Wear particles stimulate chronic inflammation that delays osseointegration, and leads to peri-prosthetic osteolysis and implant loosening. Particle-induced inflammation results in the release of pro-inflammatory factors; these events are mediated primarily by the transcription factor Nuclear Factor kappa B (NF?B), a critical signaling molecule in the activation of pro- inflammatory genes. The purpose of this grant is to modulate NF?B expression in order to mitigate particle- induced inflammation and osteolysis using in vitro and in vivo models. The research proposal will yield substantive strategies for treatment of periprosthetic osteolysis in humans. Specific Aim #1: To demonstrate that an NF?B decoy oligodeoxynucleotide (ODN) will abrogate the expression of pro-inflammatory genes and proteins when murine or human macrophages are exposed to clinically relevant polyethylene particles with/without adherent endotoxin in vitro. Specific Aim #2: To demonstrate that an NF?B decoy ODN will have no major adverse effects on murine or human osteoprogenitors when the cells are exposed to clinically relevant polyethylene particles with/without adherent endotoxin in vitro. Specific Aim #3: To demonstrate that local delivery of the NF?B decoy ODN inhibits peri-implant inflammation and osteolysis associated with continuous infusion of polyethylene particles using our validated murine femoral implant model. Specific Aim #4: To demonstrate that local delivery of the NF?B decoy ODN inhibits systemic migration of exogenous, reporter macrophages to the area of particle infusion using our murine model. Murine and human macrophages and osteoprogenitor cells (OPCs) will be cultured with clinically relevant polyethylene particles endotoxin. NF?B decoy ODN, scrambled decoy ODN or nothing will be added to the cultures. Cellular viability and proliferation will be assessed; TNF, IL-1, IL-6, MCP-1 and RANKL will be assayed at the protein and gene levels from the supernatants (ELISA and Western blot) and cells (RT- PCR) respectively. OPC cultures will be assayed for alkaline phosphatase and osteocalcin expression, and bone matrix formation will be quantitated using Von Kossa staining. Continuous infusion of polyethylene particles using our murine femoral implant model will result in systemic reporter macrophage trafficking to the site of particle infusion (using bioluminescence and immunofluorescence microscopy), increased local bone turnover (using microPET scans) and osteolysis (using quantitative microCT). These effects will be mitigated by the local delivery of NF?B decoy ODN. Polyethylene debris will continue to be generated from TJRs, even with newer polyethylenes that produce smaller, potentially more biologically active particles. The proposed research is important, innovative and clinically relevant; it will suggest potential local pharmacological strategies to mitigate the chronic inflammatory reaction to wear particles and periprosthetic osteolysis. PUBLIC HEALTH RELEVANCE: Total joint replacement (TJR) is a highly successful surgical procedure for end-stage arthritis, however the longevity of TJRs is limited by wear of the bearing surfaces. Wear particles stimulate a chronic inflammatory reaction that leads to local bone destruction around the implant. This grant application tests a novel translational strategy to mitigate particle-associated bone destruction by interfering with the primary signaling molecule (the transcription factor NFkB) within cells that controls the production of inflammatory mediators. This strategy has a high likelihood of extending the lifetime of TJRs in humans.